The invention relates to methods for simultaneously detecting both members of a binding pair in a biological sample.
Blood products used for transfusion and transfer of blood components must be routinely screened for the presence of infectious agents such as human immunodeficiency virus (HIV), hepatitis viruses, human T-lymphocytotropic virus, and cytomegalovirus. Such agents typically are detected by either identification of viral antigens or by detection of an immune response to the virus (i.e., host-derived antiviral antibodies) using enzyme immunoassay analysis (EIA) or radioimmunoassays (RIA). Immunoassay techniques are limited in their ability to detect the presence of viral contaminants in early stages of infection, with the window period between infection with a virus and detection by immunoassay techniques varying from two to four weeks for HIV and up to about 10 weeks for hepatitis C virus (HCV). Techniques such as reverse-transcriptase polymerase chain reaction (RT-PCR) or branched chain DNA analysis can shorten the time period between infection and detection, but are cost prohibitive for use on an individual donor basis and do not eliminate the window period.
The invention is based on a rapid and sensitive method for simultaneously detecting both members of a binding pair, such as a ligand and receptor or an antigen and host antibody, from a biological sample. Methods of the invention can, for example, enhance the ability to detect infections at an early stage, leading to earlier treatment of the infection.
The invention features a method for simultaneously measuring both members A and B of a binding pair in a biological sample. The biological sample is selected from the group consisting of blood, plasma, serum, urine, cerebrospinal fluid, sputum, tears, amniotic fluid, vitreous humor, saliva, and tissue culture supernatants. The method includes providing a solid phase reagent, which includes a particle coated with capture antibodies having specific binding affinities for member A of the binding pair, and contacting a biological sample with the solid phase reagent under conditions in which member A, if present, becomes bound to the particle, to form a first reacted particle. The capture antibodies can be monoclonal. The first reacted particle is contacted with first antibodies having specific binding affinities for member A, wherein the first antibodies are labeled with a first label, and with second antibodies having specific binding affinities for member B of the binding pair, wherein the second antibodies are labeled with a second label, to form a second reacted particle. The first and second antibodies can be monoclonal. First and second labels (e.g., fluorophores) are measured on the second reacted particle using flow cytometry.
In certain embodiments, substantially all capture antibodies are oriented on the particle such that the antigen binding regions of the capture antibodies are available for binding member A of the binding pair.
Member A of the binding pair can be, for example, an antigen and member B can be a host antibody. The antigen can be a viral antigen such as a hepatitis C antigen, a hepatitis B antigen, or a human immunodeficiency virus antigen, or an autoantigen such as glutarnic acid decarboxylase. Member A of the binding pair also can be a ligand, such as a cytokine, and member B can be a receptor, such as a cytokine receptor. In addition, member A can be an enzyme and member B can be a substrate. For example, the enzyme can be caspase-3 or caspase-1 and the substrate can be poly(ADP-ribose) polymerase or proInterleukin-1, respectively.
The invention also features a kit for simultaneously measuring both embers A and B of a binding pair in a biological sample. The kit includes a solid phase reagent, which includes a particle coated with capture antibodies having specific binding affinities for member A of the binding pair; first antibodies having specific binding affinities for member A of the binding pair, wherein the first antibodies are labeled with a first label; and second antibodies having specific binding affinities for member B of the binding pair, wherein the second antibodies are labeled with a second label. Substantially all the capture antibodies are oriented on the particle such that the antigen binding regions of the capture antibodies are available for binding member A of the binding pair. The kit further can include a label or package insert, which indicates that the solid phase reagent, the labeled first antibodies, and the labeled second antibodies can be used for simultaneously measuring both members A and B of a binding pair in a biological sample by flow cytometry.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.